The coordinate activities of membrane ion channels and transporters, signaling molecules, and structural proteins control cardiomyocyte excitation-contraction coupling and cardiac function. Our recent findings support a new basis for human cardiac disease based on mutations to proteins that are required for proper organization of membrane and structural proteins within the myocyte. We have found that human type 4 long QT (LQT4) syndrome results from loss-of-function mutations to ankyrin-B (AnkB), an adapter protein that links integral membrane proteins with cytoskeletal elements. Specifically, the molecular basis of atypical LQTS type 4 is an AnkB mutation (E1425G) that disrupts the binding and appropriate localization of sarcoplasmic reticulum (SR) and T-tubule channels and transporters. The objective of this grant is to examine the molecular basis for the multifaceted capabilities of AnkB in heart. Our exciting new preliminary findings demonstrate that ANK2 (encodes human AnkB) has a complex genomic structure with 52 exons that is subject to alternative transcriptional regulation. Furthermore, we identify three novel alternative mRNA splice variants that remove key exons encoding binding sites for NCX, IP3R, and the large Rho-GEF obscurin. These preliminary data support our central hypothesis that the human heart maintains a previously unidentified complement of AnkB isoforms with unique and critical functions for normal cardiac function. We hypothesize that specific ankyrin isoforms play critical roles in ion channel and transporter targeting to myocyte T-tubules and sarcoplasmic reticulum, while other isoforms are critical for organization of protein domains within the A-band. Our proposal will use a combination of innovative biochemical, imaging, cell biological, molecular, and electrophysiological methods to test our hypothesis with the following aims: Specific Aim 1: Identify and characterize novel AnkB cardiac isoforms. Specific Aim 2: Define the molecular basis for the subcellular targeting of AnkB in heart. Specific Aim 3: Define the role of novel cardiac AnkB isoforms for ion channel and transporter targeting, stability, and function in cardiomyocytes. (End of Abstract)